The present invention relates to high throughput screening assays for the identification of potential therapeutic agents and to uses of agents identified by such assays for the prevention and/or treatment of diseases related to fatty acid metabolism.
Acyl desaturase enzymes catalyze the formation of double bonds in fatty acids derived from either dietary sources or de novo synthesis in the liver. Mammals synthesize at least three distinct desaturases, which catalyze desaturation fatty acids at different positions. These enzymes are referred to by the carbon number at which the double bond is inserted, which may occur at the xcex949, xcex946 and xcex945 positions. The resulting mono-unsaturated or polyunsaturated fatty acids are substrates for incorporation into phospholipids, triglycerides, and cholesterol esters.
In human and other mammalian cells, microsomal xcex946 desaturation of the essential fatty acids linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3) is an initial and rate limiting step in the biosynthesis of both n-6 and n-3 polyunsaturated fatty acids (PUFAs). The microsomal fraction is the fraction, comprising microspheres and other structures, produced by break-up of the rough endoplasmic reticulum following ultracentrifugation and represents a readily producible form of a number of key cellular enzymes. The products of the aforementioned reactions are gammalinolenic (18:3n-6) and stearidonic acids (18:4n-3) which are subsequently elongated to dihomogammalinolenic acid (DGLA) (20:3n-6) and to the 20:4n-3 product, respectively. The resulting fatty acids are utilized as substrates of a xcex945 desaturase that generates arachidonic acid (20:4n-6) and 20:5n-3. The latter are then further elongated to 22:4n-6 and 22:5n-3, respectively, and finally used to form the products 24:4n-6 and 24:5n-3. The delta-5 desaturase that produces 20:3n9 (or Mead acid) may not be the same enzyme that produces arachidonic acid from DGLA.
In addition to the foregoing, a second microsomal xcex946 desaturation occurs on the PUFAs. The products of this desaturation, 24:5n-6 and 24:6n-3, are converted to 22:5n-6 and 22:6n-3, respectively, by peroxisomal xcex2-oxidation (Sprecher, H. (2000) Biochim. Biophys. Acta. 1486, 219-231). In addition, two sequential elongations of 20:4n-3 (i.e., 22:4n-3 and 24:4n-3) were described by Sauerwald et al. as part of putative alternative steps in the synthesis of 22:6n-3 (Sauerwald, T. U., Hachey, D. L., Jensen, C. L., Chen, H., Anderson, R. E. and Heird W. C. (1997) Pediatr. Res. 41, 183-187). These authors proposed that 24:4n-3 could undergo xcex949 desaturation to 24:5n-3 which can act as substrate of the xcex946 desaturase. Direct elongations on 18:2n-6, 18:3n-3 and 20:3n-6 are also reported.
Evidence shows that the xcex946 desaturase, which recognizes 18-carbon unsaturated fatty acids (18:2n-6 or 18:3n-3), is the same enzyme that desaturates 24-carbon substrates (De Antueno, R. J., Knickle, L C, Smith, H., Elliot, M. L., Allen, S. J., Nwaka, S., and Winther, M. D. (2001) FEBS Letters 509, 77-80, and Innis, S. M., Sprecher, H., Hachey, D., Edmond, J. and Anderson, R. E. (1999) Lipids. 34, 139-149. Cho and coworkers were the first to clone this human xcex946 desaturase and test its activity on the generally used 18-carbon PUFA substrate. (Cho, H. P., Nakamura, M. T. and Clarke, S. D. (1999) J. Biol. Chem. 274, 471-477). In addition, Sauerwald and coworkers (1997) have suggested that two xcex946 desaturation steps active on 18:3n-3 and 18:2n-6 would compete not only with each other but also with 24:5n-3 and 24:4n-6. Sprecher, in a recent review addressing this subject, has emphasized that the control of the xcex946 desaturase would be of considerable interest in animals or human studies if a single enzyme is active on 4 different fatty acids from both n-6 and n-3 families (See: Sprecher, supra).
The delta-6 desaturase carries out desaturation on at least 4 distinct substrates: 18:3n6, 24:4n6, 18:3n3 and 24:5n3. See: De Antueno R J, Knickle L C, Smith H, Elliott M L, Allen S J, Nwaka S, Winther M D, xe2x80x9cActivity of human Delta5 and Delta6 desaturases on multiple n-3 and n-6 polyunsaturated fatty acid,xe2x80x9d FEBS Lett. Nov 30;509(1):77-80 (2001); D""andrea S, Guillou H, Jan S, Catheline D, Thibault J N, Bouriel M, Rioux V, Legrand P., xe2x80x9cThe same rat Delta6-desaturase not only acts on 18- but also on 24-carbon fatty acids in very-long-chain polyunsaturated fatty acid biosynthesis,xe2x80x9d Biochem J 364(Pt1):49-55 (May 15, 2002). In addition, 18:1n9 (oleic acid) should also be a substrate (although, perhaps, with lower affinity).
There are several different elongases present in mammalian cells, which are active on different fatty acid substrates. Some of these have been characterized (see Winther et al, WO 02/44320). In at least one example a genetic defect in a human elongase is associated with a human disease, indicating the importance of this enzyme family as a therapeutic target (Zhang, K., Kniazeva, M. Han, M. et al (2001) Nature Genetics 27, 89-93).
It has been shown (see Brownlie et al, WO 01/62954) that delta-9 desaturase activity may be indirectly measured in a microsomal assay by measuring the release of tritium from the C9 and C10 positions of stearoyl-CoA, in the form of water. In accordance with the present invention, this kind of assay is applicable to a wide range of other desaturase and elongase enzymes using an efficient and economical tritium based assay. Using the process disclosed herein, modulators of fatty acid or triglyceride metabolism are readily identified.
In one aspect, the present invention relates to a process for identifying a desaturase-modulating agent, comprising:
a) contacting a compound with a microsome, said microsome comprising a desaturase enzyme activity, in the presence of a substrate comprising a tritium label wherein said label is bound to a desaturase reactive carbon atom and under conditions promoting the desaturation of said substrate at said reactive carbon atom to form a double bond with said reactive carbon atom with release of tritiated water,
b) detecting production of said tritiated water,
c) detecting a difference in the amount or rate of production of said tritiated water compared to when the compound of step (a) is not present thereby identifying a modulator of desaturase activity.
In specific embodiments thereof, the modulation may be an increase or a decrease in desaturase activity and may be determined as either a difference in the amount of said tritiated water detected and/or a difference in the rate of production of said tritiated water. In addition, the tritium label may be present on one or both of the desaturase reactive carbon atoms (i.e., the carbon atoms forming the double bond resulting from this desaturation reaction). In preferred embodiments, the desaturase is a delta-5 desaturase or a delta-5 desaturase.
Preferred substrates for delta-5 desaturases include 20 carbon backbone fatty acids, such as a coenzyme A conjugate of dihomogammalinolenic acid (20:3n6, DGLA), 20:4n3 and 20:2n9, especially where the tritium label is on carbon atom 5 and 6 of the substrate.
Preferred substrates for the delta-6 desaturase include a substrate that is a coenzyme A conjugate of a fatty acid having a backbone of 18 carbon atoms or 24 carbon atoms in length, preferably a coenzyme A conjugate of xcex1-linolenic acid (18:3n3), 24:5n3, linoleic acid (18:2n6), 18:1n9 or 24:4n6, especially where the tritium label is on carbon atom 6 and 7 of the substrate.
In another aspect, the present invention relates to a process for identifying an elongase- and/or desaturase-modulating agent, comprising:
a) contacting a compound with a microsome, said microsome having a desaturase enzyme activity, in the presence of an elongase and a substrate of said elongase, wherein said substrate is not a substrate of said desaturase enzyme activity, under conditions promoting elongation of said substrate to form an elongated product wherein said product is a substrate of said desaturase and comprises a tritium label bound to a desaturase reactive carbon atom and under conditions promoting desaturation of said substrate of said desaturase to form a double bond involving said desaturase reactive carbon atom with release of tritiated water,
b) detecting production of said tritiated water,
c) detecting a difference in the amount or rate of production of said tritiated water compared to when the compound of step (a) is not present, thereby identifying a modulator of elongase and/or desaturase activity.
Preferred embodiments include wherein said modulation is a decrease or increase in desaturase and/or elongase activity, preferably where the desaturase is a delta-5 desaturase and where the substrate for the elongase is a 16, 18, 20 or 22-carbon atom backbone fatty acid.
Substrates of the elongase may also be substrates of the n9 pathway (which starts with oleic acid 18:1n9) and the n7 pathway (which starts with palmitic acid (16:0). Elongation substrates would be: 18:2n9, 20:3n9, 18:1n9, 20:1n9, 22:1n9, 16:0, 16:1n7, 18:1n7, 20:1n7.
In a further aspect, the present invention relates to a process for identifying an elongase-modulating agent, comprising:
a) contacting a compound with a microsome, said microsome having a desaturase enzyme activity, in the presence of an elongase and a substrate of said elongase, wherein said compound is not a modulator of said desaturase activity using the process disclosed herein without the elongase for determining modulators of desaturase activity and wherein said substrate is not a substrate of said desaturase enzyme activity, under conditions promoting elongation of said substrate to form an elongated product wherein said product is a substrate of said desaturase and comprises a tritium label bound to a desaturase reactive carbon atom and under conditions promoting desaturation of said substrate of said desaturase to form a double bond involving said desaturase reactive carbon atom with release of tritiated water,
b) detecting production of said tritiated water,
c) detecting a difference in the amount or rate of production of said tritiated water compared to when the compound of step (a) is not present, and thereby identifying a modulator of elongase activity.
Preferred embodiments include wherein said modulation is a decrease or increase in desaturase and/or elongase activity, preferably where the desaturase is a delta-5 desaturase and where the substrate for the elongase is an 18, 20 or 22-carbon atom backbone fatty acid.
Substrates of the elongase again would include substrates of the n9 pathway (which starts with oleic acid 18:1n9) and the n7 pathway (which starts with palmitic acid (16:0). Elongation substrates would be: 18:2n9, 20:3n9, 18:1n9, 20:1n9, 22:1n9,16:0, 16:1n7, 18:1n7, 20:1n7.
In an additional aspect, the present invention relates to a process for identifying a therapeutic agent useful in modifying fatty acid levels to bring about therapeutic interventions in a range of diseases, comprising identifying an agent according to the modulator-determining processes of the invention and further comprising administering said agent to an animal and then detecting a decrease in said triglyceride levels following said administration thereby identifying such a therapeutic agent. Preferred embodiments thereof include where the animal is afflicted with metabolic disorders with altered fatty acid metabolism, most preferably where said animal is a human being.
In a still further aspect, the present invention relates to a process for treating a disease of fatty acid metabolism in an animal comprising administering to an animal afflicted therewith a therapeutically effective amount of a compound first identified as a desaturase and/or elongase modulating agent using an assay as disclosed herein according to the invention, especially where said animal is a human being.
In a yet further aspect, the present invention relates to a process for protecting an animal against a disorder involving fatty acid metabolism comprising administering to an animal predisposed to developing such a disorder a therapeutically effective amount of a compound first identified as a desaturase and/or elongase modulating agent using an assay of the invention, preferably where said animal is a human being, most preferably a human genetically predisposed to develop said disorder.
In a yet still further aspect, the present invention relates to a method for producing a product comprising identifying an agent according to any of the assay processes of the invention wherein said product is the data collected with respect to said agent as a result of said process and wherein said data is sufficient to convey the chemical structure and/or properties of said agent.